Abstract

The p57KIP2 is a maternally expressed and
paternally imprinted cyclin-dependent kinase inhibitor located on
chromosome 11p15.5. Because of its location, biochemical functions, and
imprinting status, p57KIP2 has been
considered a candidate tumor suppressor gene. To determine, for the
first time, the involvement of this gene in the development of head and
neck squamous carcinoma (HNSC), we analyzed the imprinting and
expression status and loss of heterozygosity (LOH) within the
p57KIP2 gene flanking loci on the 11p15.5
region in 64 primary untreated tumors. Of the 30 (47%) informative
cases for this gene, loss of imprinting and LOH were noted in 4 (13%)
and 10 tumors (33%), respectively. Analysis of the microsatellite
markers flanking the p57KIP2 gene on
chromosome 11p showed infrequent alterations at these loci.
p57KIP2 was expressed in all tumors with LOH
within and around the gene. Quantitative reverse transcription-PCR
analysis showed elevated p57 mRNA expression in tumor
with loss of imprinting. Sequencing analysis of exons 1 and 2 of the
p57KIP gene failed to detect any mutations.
Our data indicate: (a) infrequent genomic abnormalities
at the p57KIP2 gene in HNSC;
(b) leaky or incomplete imprinting of the paternal
allele is associated with increased expression of this gene in a subset
of tumors; and (c) minimal evidence for suppressor
function for this gene in HNSC.

INTRODUCTION

Genomic imprinting is the epigenetic marking of a gene, based on
parental origin, that results in monoallelic expression
(1, 2, 3, 4)
. This phenomenon differs from the classical
sequence-based qualitative changes in that gene expression and
effective gene dosage are controlled by epigenetic dysregulation of
parental alleles of an imprinted gene. Imprinting dysregulation may
contribute to tumorigenesis either by activating a transcriptionally
repressed allele resulting in gene activation, or by inactivating an
expressed allele of an imprinted tumor suppressor gene, leading to loss
of function (5)
. Evidence implicating this process in
tumorigenesis is based on the finding of selective parental
LOH3
and LOI at certain imprinted domains in several pediatric tumors
(6, 7, 8, 9, 10, 11, 12, 13)
.

The chromosome 11p15.5 region contains imprinted domains in which
H19,IGF2, and the recently identified
p57KIP2 genes reside
(14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25)
. Several studies have shown that imprinting
abnormalities of the H19 and/or IGF2 genes play a
role in tumorigenesis of both embryonal and adult neoplasms (14, 18, 19, 20, 21, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36)
. p57KIP1, a
maternally expressed gene located ∼400–500 kb centromeric to
H19 and IGF2 genes, is a member of INK’s CDK
inhibitor family, which includes p21CIP1
and p27KIP1 genes (13, 14, 15, 22)
. A tumor suppressor role for the
p57KIP2 has been suggested in studies of
different tumors based on its association with cell cycle control and
imprinting status. Therefore, the dysregulation of
p57KIP2 by imprinting of the maternally
expressed allele, or by relaxation of this process in the paternal
allele, results in uncontrolled proliferation and tumor development
(3, 9, 17, 18, 21, 22, 36, 37)
.

We investigated, for the first time, the incidence of imprinting and
genetic alterations at the p57 gene and its flanking loci on
chromosome arm 11p15.5 region in 64 primary oral squamous carcinoma to
determine their association with head and neck squamous tumorigenesis.

MATERIALS AND METHODS

Specimens.

The materials for this study consisted of matched pairs of fresh normal
squamous mucosa and tumor specimens, which were surgically removed from
64 patients with primary oral squamous cell carcinoma between
1993 and 1996 at the Department of Pathology, The University of Texas
M. D. Anderson Cancer Center (Houston, TX). All specimens were
received by one pathologist (A. E. N.) and were subjected to frozen
section evaluation and stored at −80°C until used. Normal mucosae
from the same patients were obtained from the farthest margin of
resection after frozen section verification.

DNA and RNA Extraction.

DNA and RNA isolation were performed according to previously published
protocol (38)
.

Characterization of p57 and Chromosome 11p
Polymorphism.

Primers C and D (Table 1)⇓
were used to PCR amplify normal and tumor genomic DNA to determine the
heterozygosity of p57(21)
. The reaction
mixture was composed of 1× PCR buffer (Promega Corp., Madison, WI)
with 1 μm of primers, 2000 μm of deoxynucleotide triphosphate, 2.5
units of Taq polymerase, and 100 ng of template DNA. PCR conditions
were 98°C for 5 min, 35 cycles of 95°C for 1 min, 65°C for
30 s, and 72°C for 30 s, followed by a 72°C 5-min
extension.

Two microsatellite markers, THO1 and
D11S2359 (Research Genetics, Inc., Huntsville, AL), flanking
p57 loci on 11p15.5 were also used to detect the
heterozygosity of 11p. PCR products were separated in 10% acrylamide
gel and stained using a color silver staining kit (Pierce Chemical Co.,
Rockford, IL).

RT-PCR.

For allele-specific expression, 1 μg of total RNA from normal mucosa
and tumor specimens was reverse-transcribed for the first strand of
cDNA using the Gene Amp RT-PCR system (Perkin-Elmer Cetus,
Branchburg, NJ) in a 20-μl reaction. The reaction mixture was
added to 80 μl of 100 μm deoxynucleotide triphosphate
and 2 mm MgCl2, 10% glycerol, and
2.5 units of Taq polymerase in 1× PCR buffer. PCR was carried out with
0.4 μm of primer E1 and D (Table 1)⇓
. The PCR
condition for primers E1 and D was similar to
those of primers C and D, except that annealing was performed at 60°C
for 1 min and extension was performed at 72°C for 1 min. This
resulted in a 547-bp p57 cDNA band, whereas primers
E1 and D yielded a 668-bp DNA fragment (data not
shown) and served as a size reference (21)
on 2% agarose
gel.

QC-RT-PCR was performed with the PCR mimic construction kit
(Clontech Industries Inc., Palo Alto, CA). Primers 11 and 12 consisted
of the flanking nucleotide sequence of p57 cDNA (Table 1)⇓
.
PCR amplification of the v-erb fragment as a competitor was
developed using the same primer (11 and 12) sequences in addition to
fragments A and B of the v-erb gene. Thus, primers 11 and 12
were used to coamplify the 1:10 dilution of the competitor and
p57 cDNA under the same PCR conditions. Products were run on
2% agarose gel stained with ethidium bromide, and band density was
calibrated by a densitometer (Molecular Dynamics, Sunnyvale, CA). The
level of p57 mRNA was determined by comparing the intensity ratio of
competitor:target bands.

Imprinting Analysis.

In heterozygous cases for the p57 gene, primers C and D were
used for PCR from the 547-bp cDNA template of normal and tumor samples
to obtain a nested product, which was run in 6% acrylamide gel viewed
by silver staining (Pierce Chemical Co.). This nested PCR product from
the normal sample was also run in parallel with normal and tumor
genomic DNA PCR product by primers C and D.

SSCP Analysis.

Mutation screening of exons 1 and 2 was carried out by SSCP analysis.
Exon 1 was amplified as one fragment with primers 1U and 1D, whereas
exon 2 was split into three fragments with primers 2AU and 2AD, 2BU and
2BD, and 2CU and 2CD for amplification (Table 1)⇓
. Five μl of PCR
products were denatured by heating at 95°C for 5 min in 5 μl of
sequencing stop solution (United States Biochemical Corp., Cleveland,
OH). The reaction mixture was applied to 8% nondenaturing acrylamide
gel at 150 V overnight at 4°C. The gel was subjected to silver
staining using a color silver stain kit (Pierce Chemical Co.).

RESULTS

Of the 64 tumors analyzed, 30 (47%) were heterozygous for the
p57 gene (Table 2)⇓
, with two bands of 103- and 91-bp PCR product identified in normal
specimen (data not shown). These were considered informative for
further analysis. Materials from these cases were used to investigate
the LOH at the p57KIP2 polymorphic locus in
exon 2 (DNA) and the imprinting abnormalities of this gene (RNA).
Constitutive imprinting yielded a band of either 103 or 91 bp. Four of
the heterozygous tumors (13%) manifested LOI, as evidenced by the
biallelic expression of both the 103-bp and 91-bp alleles of the
p57 gene on cDNA in tumor samples, whereas corresponding
normal mucosa showed monoallelic expression (Fig. 1⇓
, cases 45 and 62). In QC-RT-PCR, the 1:1 intensity ratio of
competitor:p57 cDNA represented a p57 concentration equal to that of
the competitor. Tumor with LOI showed increased RNA expression compared
with matched normal specimens (Fig. 2)⇓
, whereas no detectable changes were noted in tumor lacking imprinting
or genetic alterations (cases 5 and 22).

QC-RT-PCR analysis of the p57 mRNA level in a
case (45)
with LOI. Serial dilutions of the competitor
(v-erb) were coamplified with either normal or tumor cDNA samples. The
gel photograph represents competitor (C) PCR products
with normal (N; top) and tumor
(T; bottom) cDNA samples. The
intensity ratio of competitor:normal as well as competitor:tumor p57
mRNA was plotted in relation to the log scale of competitor
concentration. At the ratio of 1, tumor concentration is higher than
that of the normal specimen.

LOH was scored if one band showed a >50% intensity reduction in tumor
specimen. LOH at the p57 polymorphic locus on exon 2 was noted in 10
(33.3%) tumors (Fig. 3⇓
, cases 47 and 61; Table 2⇓
). Analysis of the microsatellite markers
flanking the p57KIP2 locus on chromosome
11p15.5 showed LOH in six tumors (13%) of the 45 informative cases at
marker D11S2359 and in seven tumors (14%) of the
informative cases at marker THO1 (data not shown). One tumor
specimen showed instability at the THO1 marker. Of the 27
(45%) informative cases for both the p57 polymorphic locus and markers
D11S2359 and THO1, two tumors (7%) showed
simultaneous LOH at the p57 locus and either one of the
microsatellite markers, and one tumor manifested LOH at the
p57 locus and instability of the THO1 locus. None
of the tumors manifested concurrent LOI and LOH. No correlation between
LOI and LOH at the p57 gene or the flanking microsatellite
markers was noted (P = 0.61; Table 3⇓
).

Correlation between p57 gene
alterations and LOH at its flanking microsatellite markers in HNSC

Our mutational screening of the p576 exons by SSCP analysis failed to
yield any mutations in exons 1 and 2. However, a polymorphism
representing a 12-bp deletion in one allele was observed. To determine
preferential parental allelic loss, comparison of the banding pattern
between cDNA of normal mucosa and normal and tumor DNA from the 10
cases with LOH at the p57KIP2 gene were
performed. Loss of maternal allele was considered if the same DNA band
that appeared in normal cDNA was lost (assuming that the maternal
allele is expressed in normal cDNA). We observed equal allelic loss at
both alleles of the p57 gene, indicating lack of specific
paternal allelic loss of this gene (Fig. 4⇓
, cases 18, 39, 47, and 61).

Comparative DNA and cDNA for preferential loss
of a parental allele: Variable parental allelic loss in DNA and cDNA
from cases 47 and 61 as well as cases 18 and 39 is noted, indicating
lack of preferential parental LOH.

Our results show infrequent alterations of the p57KIP2 gene
and detectable expression in all cases with LOH within and at the
flanking 11p15.5 loci of this gene in HNSC. We also observed biallelic
expression and elevation of mRNA content in tumors with LOI. These
results, together with the lack of sequence alterations and the
consistent expression, argue against a tumor suppressor role for this
gene in HNSC (3, 9, 11, 17, 22, 36, 37, 39, 40, 41)
.
Our results, however, are in agreement with previous studies of
embryonal and lung carcinomas (10, 16, 42, 43)
and are
supported by in vitro experiments negating an association
between p57KIP2 and tumor suppression
(12)
. Paradoxically, a tumor suppressor function is
evinced by the detection of nonsense mutation in its CDK inhibitory
domain, development of Beckwith-Wiedemann syndrome phenotype in
knockout mice, lack of expression in certain tumors, and association of
G1 cell arrest with overexpression (9, 14, 44, 45, 46, 47)
. The differences between these studies could be
attributed to organ- or tumor-specific modifications of this gene.

Our findings of expression in cases with LOH at the p57 gene
in our case are similar to those previously reported in lung carcinoma
(39)
. Contrary to the lung carcinoma study
(39)
, no selective parental LOH of the
p57Ink4 in our cases was observed. These
results suggest that variations in the imprinting level account for the
p57 expression level in tumors. Quantitative studies to precisely
determine the effect of imprinting status on the expression level of
this gene are needed. Taken together, the lack of preferential parental
loss, infrequent imprinting dysregulation, and rare genetic alterations
indicate a minimal role for the p57 gene in HNSC. However,
the biallelic and elevated mRNA expression in some of our cases and in
other tumor subtypes (22, 45)
indicate either a
leaky/incomplete imprinting or paternal LOI and suggest a
possible oncogenic role for this gene in a subset of these tumors.
Further studies are needed to determine the biological effect of the
biallelic expression of this gene in tumorigenesis.

Footnotes

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.